Exploration of a multifunctional biocontrol agent Streptomyces sp. JCK-8055 for the management of apple fire blight.

Apple fire blight, caused by the bacterium Erwinia amylovora, is a devastating disease of apple and pear trees. Biological control methods have attracted much attention from researchers to manage plant diseases as they are eco-friendly and viable alternatives to synthetic pesticides. Herein, we isolated Streptomyces sp. JCK-8055 from the root of pepper and investigated its mechanisms of action against E. amylovora. Streptomyces sp. JCK-8055 produced aureothricin and thiolutin, which antagonistically affect E. amylovora. JCK-8055 and its two active metabolites have a broad-spectrum in vitro activity against various phytopathogenic bacteria and fungi. They also effectively suppressed tomato bacterial wilt and apple fire blight in in vivo experiments. Interestingly, JCK-8055 colonizes roots as a tomato seed coating and induces apple leaf shedding at the abscission zone, ultimately halting the growth of pathogenic bacteria. Additionally, JCK-8055 can produce the plant growth regulation hormone indole-3-acetic acid (IAA) and hydrolytic enzymes, including protease, gelatinase, and cellulase. JCK-8055 treatment also triggered the expression of salicylate (SA) and jasmonate (JA) signaling pathway marker genes, such as PR1, PR2, and PR3. Overall, our findings demonstrate that Streptomyces sp. JCK-8055 can control a wide range of plant diseases, particularly apple fire blight, through a combination of mechanisms such as antibiosis and induced resistance, highlighting its excellent potential as a biocontrol agent. KEY POINTS: • JCK-8055 produces the systemic antimicrobial metabolites, aureothricin, and thiolutin. • JCK-8055 treatment upregulates PR gene expression in apple plants against E. amylovora. • JCK-8055 controls plant diseases with antibiotics and induced resistance.

Elucidation of the nematicidal mode of action of grammicin on Caenorhabditis elegans.

Grammicin (Gra) is derived from the endophytic fungus Xylaria grammica EL000614 and shows nematicidal activity against the devastating root-knot nematode Meloidogyne incognita in-vitro, in planta, and in-field experiments. However, the mechanism of the nematicidal action of Gra remains unclear. In this study, Gra exposure to the model genetic organism Caenorhabditis elegans affected its L1, L2/3, L4, and young adult stages. In addition, Gra treatment increased the intracellular reactive oxygen species (ROS) levels of C. elegans and M. incognita. Molecular docking interaction analysis indicated that Gra could bind and interact with GCS-1, GST-4, and DAF-16a in order of low binding energy, followed by SOD-3, SKN-1, and DAF-16b. This implies that the anthelmintic action of Gra is related to the oxidative stress response. To validate this mechanism, we examined the expression of the genes involved in the oxidative stress responses following treatment with Gra using transgenic C. elegans strains such as the TJ356 strain zIs356 [daf-16p::daf-16a/b::GFP + rol-6 (su1006)], LD1 ldIs7 [skn-1p::skn-1b/c::GFP + rol-6 (su1006)], LD1171 ldIs3 [gcs-1p::GFP + rol-6 (su1006)], CL2166 dvIs19 [(pAF15) gst-4p::GFP::NLS], and CF1553 strain muIs84 [(pAD76) sod-3p::GFP + rol-6 (su1006)]. Gra treatment caused nuclear translocation of DAF-16/FoxO and enhanced gst-4::GFP expression, but it had no change in sod-3::GFP expression. These results indicate that Gra induces oxidative stress response via phase II detoxification without reduced cellular redox machinery. Gra treatment also inhibited the nuclear localization of SKN-1::GFP in the intestine, which may lead to a condition in which oxidative stress tolerance is insufficient to protect C. elegans by the inactivation of SKN-1, thus inducing nematode lethality. Furthermore, Gra caused the mortality of two mutant strains of C. elegans, CB113 and DA1316, which are resistant to aldicarb and ivermectin, respectively. This indicates that the mode of action of Gra is different from the traditional nematicides currently in use, suggesting that it could help develop novel approaches to control plant-parasitic nematodes.

Inhibition of Oomycetes by the Mixture of Maleic Acid and Copper Sulfate.

Since the protective activity of the Bordeaux mixture against plant disease caused by oomycetes was discovered, copper compounds have been used for more than a century as an effective plant protection strategy. However, the application of excessive copper can cause adverse effects through long-term heavy metal accumulation in soils. Therefore, it is necessary to develop new strategies to reduce or replace copper in pesticides based on organic and low-input farming systems. Organic acids are eco-friendly. In this study, we tested the antifungal and anti-oomycete activity of maleic acid (MA) and copper sulfate (CS) against 13 plant pathogens. Treatment with a mixture of MA and CS showed strong anti-oomycetes activity against Phytophthora xcambivora, P. capsici, and P. cinnamomi. Moreover, the concentration of CS in the activated mixture of MA and CS was lower than that in the activated CS only, and the mixture showed synergy or partial synergy effects on the anti-oomycete activity. Application of a wettable powder formulation of MA and CS mixture (MCS 30WP; 26.67% MA and 3.33% CS) had excellent protective activity in pot experiments with control values of 73% Phytophthora blight on red pepper, 91% damping-off on cucumber, and 84% Pythium blight on creeping bentgrass, which are similar to those of the CS wettable powder formulation (6.67% CS) containing two times the CS content of MCS 30WP. These observations suggest that the synergistic effect of the MA and CS combination is a sustainable alternative for effective management of destructive oomycete diseases.

First Report of Shot-Hole on Flowering Cherry Caused by Burkholderia contaminans and Pseudomonas syringae pv. syringae.

Shot-hole disease (SH) is one of the most common and important diseases affecting flowering cherry (FC; Prunus × yedoensis Matsumura; Somei-yoshino) trees in South Korea every year, resulting in premature defoliation and reduced flowering in the following year. However, pathogens associated with the disease remain unknown, which has rendered disease management challenging. Here, the pathogens associated with SH, their biochemical characteristics, and their host range were elucidated. Detached-leaf and in planta assays revealed that two biofilm-forming bacteria-namely, Burkholderia contaminans and Pseudomonas syringae pv. syringae-caused SH of FC trees. These pathogens were recorded for the first time as the causes of SH of FC trees in South Korea. Additionally, the two pathogens induced similar disease symptoms in several stone fruit belonging to the genus Prunus, including peach (Prunus persica), plum (P. salicina), and apricot (P. mume), with peach being the most susceptible. These results indicate that B. contaminans and P. syringae pv. syringae caused SH on FC trees and presented a broad spectrum of hosts. Furthermore, Xanthomonas arboricola pv. pruni, the causative agent of leaf spot on stone fruit, incited brown spots and shot holes on FC leaves. Therefore, FC trees are susceptible to infections by various pathogenic bacteria, including B. contaminans, P. syringae pv. syringae, and X. arboricola pv. pruni. These findings will be of great importance as a reference for effective management of SH in the face of possible cross-infection between Prunus spp. in the future.

Nematicidal Activity of Grammicin Biosynthesis Pathway Intermediates in Xylaria grammica KCTC 13121BP against Meloidogyne incognita.

Grammicin, a polyketide metabolite produced by the endolichenic fungus Xylaria grammica KCTC 13121BP, shows strong nematicidal activity against Meloidogyne incognita. This study was performed to elucidate the grammicin biosynthesis pathway of X. grammica KCTC 13121BP and to examine the nematicidal activity of the biosynthesis intermediates and derivatives against M. incognita. Two grammicin biosynthesis intermediates were isolated from a T-DNA insertion transformant (strain TR-74) of X. grammica KCTC 13121BP and identified as 2-(hydroxymethyl)cyclohexa-2,5-diene-1,4-dione (compound 1) and 2,5-dihydroxybenzaldehyde (compound 2), which were also reported to be intermediates in the biosynthesis pathway of patulin, an isomer of grammicin. This indicates that the grammicin biosynthesis pathway overlaps almost with that of patulin, except for the last few steps. Among 13 grammicin biosynthesis intermediates and their derivatives (except grammicin), toluquinol caused the highest M. incognita J2 mortality, with an LC50/72 h value of 11.13 µg/mL, which is similar to grammicin with an LC50/72 h value of 15.95 µg/mL. In tomato pot experiments, the wettable powder type formulations (WP) of toluquinol (17.78 µg/mL) and grammicin (17.78 µg/mL) also effectively reduced gall formation on the roots of tomato plants with control values of 72.22% and 77.76%, respectively, which are much higher than abamectin (16.67%), but lower than fosthiazate (100%). The results suggest that toluquinol can be used directly as a biochemical nematicide or as a lead molecule for the development of new synthetic nematicides for the control of root-knot nematode diseases.

Greenness, civil environment, and pregnancy outcomes: perspectives with a systematic review and meta-analysis.

BACKGROUND: Various maternal conditions, especially in utero conditions and prenatal exposure to environments with air pollution and greenness, have been reviewed to address the enhancement and prevention of susceptibility to health risks, including low birthweight, preterm delivery, and preeclampsia. This study aimed to qualitatively and quantitatively investigate the associations between pregnancy outcomes and the characteristics of surrounding living environment, including greenness, air pollution, and civilization. METHODS: A secondary search of the MEDLINE, EMBASE, Cochrane Library, K-eArticles, and CINAHL databases was conducted without language restrictions to identify the relevant publications from the time of inception of the databases to April 2019. RESULTS: A total of 89 studies were identified, and 10 were included in the quantitative synthesis. The greenness of the environment within 100-, 250- and 500-m buffers, after adjusting for the air quality and civilization factors, was weakly but positively associated with birthweight. The pooled regression slope was 0.00134 (95% confidence interval [CI], 0.000, 0.0020). The greenness of the environment was also associated with a significant decrease in the incidence of poor pregnancy outcomes, namely, low birthweight, small for gestational age (odds ratio [OR] 0.94; 95% CI, 0.92, 0.97), and preterm delivery (OR 0.98; 95% CI, 0.97, 0.99). CONCLUSIONS: The greenness of the environment had a positive effect on the pregnancy outcomes, despite poor air quality and civilization. Following urbanization, planning for greenness management, environmental medicine, and public health is important and thus should be proposed as preventive methods as way of increasing birthweight and life expectancy.

Genome-wide exonic small interference RNA-mediated gene silencing regulates sexual reproduction in the homothallic fungus Fusarium graminearum.

Various ascomycete fungi possess sex-specific molecular mechanisms, such as repeat-induced point mutations, meiotic silencing by unpaired DNA, and unusual adenosine-to-inosine RNA editing, for genome defense or gene regulation. Using a combined analysis of functional genetics and deep sequencing of small noncoding RNA (sRNA), mRNA, and the degradome, we found that the sex-specifically induced exonic small interference RNA (ex-siRNA)-mediated RNA interference (RNAi) mechanism has an important role in fine-tuning the transcriptome during ascospore formation in the head blight fungus Fusarium graminearum. Approximately one-third of the total sRNAs were produced from the gene region, and sRNAs with an antisense direction or 5'-U were involved in post-transcriptional gene regulation by reducing the stability of the corresponding gene transcripts. Although both Dicers and Argonautes partially share their functions, the sex-specific RNAi pathway is primarily mediated by FgDicer1 and FgAgo2, while the constitutively expressed RNAi components FgDicer2 and FgAgo1 are responsible for hairpin-induced RNAi. Based on our results, we concluded that F. graminearum primarily utilizes ex-siRNA-mediated RNAi for ascosporogenesis but not for genome defenses and other developmental stages. Each fungal species appears to have evolved RNAi-based gene regulation for specific developmental stages or stress responses. This study provides new insights into the regulatory role of sRNAs in fungi and other lower eukaryotes.

Biological control of tomato bacterial wilt by oxydifficidin and difficidin-producing Bacillus methylotrophicus DR-08.

Bacillus methylotrophicus DR-08 exhibited strong antibacterial activity against Ralstonia solanacearum, a causal agent of tomato bacterial wilt. This study aimed to identify the antibacterial metabolites and evaluate the efficacy of the strain as a biocontrol agent for tomato bacterial wilt. A butanol extract of the DR-08 broth culture completely inhibited the growth of 14 phytopathogenic bacteria with minimum inhibitory concentration (MIC) values of 1.95-500 µg/mL. R. solanacearum was highly sensitive to the DR-08 extract, with an MIC value of 12.62 µg/mL. Two antibacterial metabolites were isolated and identified as difficidin and oxydifficidin derivatives through bioassay-guided fractionation and instrumental analyses. Both metabolite derivatives inhibited the growth of most of the phytopathogenic bacteria tested and the oxydifficidin derivatives generally presented a stronger antibacterial activity than the difficidin derivatives. A 30% suspension concentrate of DR-08, at a 500-fold dilution, effectively suppressed the development of tomato bacterial wilt in pot and field experiments. It also effectively reduced the development of bacterial leaf spot symptoms on peach and red pepper. The results of this study suggests that B. methylotrophicus DR-08 can be utilized as a biocontrol agent for various bacterial plant diseases including tomato bacterial wilt.

A Fungus-Inducible Pepper Carboxylesterase Exhibits Antifungal Activity by Decomposing the Outer Layer of Fungal Cell Walls.

Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.

Autoregulation of ZEB2 expression for zearalenone production in Fusarium graminearum.

Several Fusarium species produce the polyketide mycotoxin zearalenone (ZEA), a causative agent of hyperestrogenic syndrome in animals that is often found in F. graminearum-infected cereals in temperate regions. The ZEA biosynthetic cluster genes PKS4, PKS13, ZEB1 and ZEB2 encode a reducing polyketide synthase, a non-reducing polyketide synthase, an isoamyl alcohol oxidase and a transcription factor respectively. In this study, the production of two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum via an alternative promoter was characterized. ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggers the induction of both ZEB2L and ZEB2S transcription. ZEB2L and ZEB2S interact with each other to form a heterodimer that regulates ZEA production by reducing the binding affinity of ZEB2L for the ZEB2L gene promoter. Our study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production; this regulatory mechanism is similar to that observed in higher eukaryotes.

Fss1 is involved in the regulation of an ENA5 homologue for sodium and lithium tolerance in Fusarium graminearum.

Sodium is an abundant cation required for protein function and maintenance of cellular osmotic homeostasis. High concentrations of sodium are toxic, and fungi have evolved efficient sodium efflux systems. In this study, we characterized a novel sodium tolerance mechanism in the plant pathogen Fusarium graminearum. Fusarium graminearum sodium sensitive 1 (Fss1) is a nuclear transcription factor with a Zn(II)2 Cys6 fungal-type DNA-binding domain required for sodium tolerance. RNA-seq and genetic studies revealed that a P-type ATPase pump, exitus natru (Latin: exit sodium) 1 (FgEna5), mediates the phenotypic defects of FSS1 mutants. A homologue of PACC (PAC1) was required for FgEna5-dependent sodium and lithium tolerance independent of Fss1. The results of this study revealed that F. graminearum has a distinct and novel pathway for sodium tolerance not present in other model fungi.

ELP3 is involved in sexual and asexual development, virulence, and the oxidative stress response in Fusarium graminearum.

Fusarium graminearum is an important fungal plant pathogen that causes serious losses in cereal crop yields and mycotoxicoses in humans and livestock. In this study, we characterized an insertion mutant, Z39R9282, with pleiotropic defects in sexual development and virulence. We determined that the insertion occurred in a gene encoding an ortholog of yeast elongator complex protein 3 (ELP3). Deletion of elp3 led to significant defects in sexual and asexual development in F. graminearum. In the elp3 deletion mutant, the number of perithecia formed was reduced and maturation of perithecia was delayed. This mutant also produced morphologically abnormal ascospores and conidia. Histone acetylation in the elp3 deletion mutant was reduced compared with the wild type, which likely caused the developmental defects. Trichothecenes were not produced at detectable levels, and expression of trichothecene biosynthesis genes were significantly reduced in the elp3 deletion mutant. Infection of wheat heads revealed that the elp3 deletion mutant was unable to spread from inoculated florets to neighboring spikelets. Furthermore, the elp3 deletion mutant was more sensitive to oxidative stress than the wild type, and the expression of putative catalase genes was reduced. We demonstrate that elp3 functions in sexual and asexual development, virulence, and the oxidative stress response of F. graminearum by regulating the expression of genes involved in these various developmental processes.

Functional characterization of sucrose non-fermenting 1 protein kinase complex genes in the Ascomycete Fusarium graminearum.

Sucrose non-fermenting 1 (SNF1) protein kinase complex is a heterotrimer that functions in energy homeostasis in eukaryotes by regulating transcription of glucose-repressible genes. Our previous study revealed that SNF1 of the homothallic ascomycete fungus Fusarium graminearum plays important roles in vegetative growth, sexual development, and virulence. In this study, we further identified the components of the SNF1 complex in F. graminearum and characterized their functions. We found that the SNF1 complex in F. graminearum consists of one alpha subunit (FgSNF1), one beta subunit (FgGAL83), and one gamma subunit (FgSNF4). Deletion of Fggal83 and Fgsnf4 resulted in alleviated phenotype changes in vegetative growth and sexual development as compared to those of the Fgsnf1 deletion mutant. However, all of the single, double, and triple deletion mutants among Fgsnf1, Fggal83, and Fgsnf4 had similar levels of decreased virulence. In addition, there was no synergistic effect of the mutant (single, double, or triple deletions of SNF1 complex component genes) phenotypes except for sucrose utilization. In this study, we revealed that FgSNF1 is mainly required for SNF1 complex functions, and the other two SNF1 complex components have adjunctive roles with FgSNF1 in sexual development and vegetative growth but have a major role in virulence in F. graminearum.

Biocontrol of Fusarium head blight in rice using Bacillus velezensis JCK-7158.

Fusarium head blight (FHB) is a destructive disease caused by several species of Fusarium, such as Fusarium graminearum and F. asiaticum. FHB affects cereal crops, including wheat, barley, and rice, worldwide. Fusarium-infected kernels not only cause reduced yields but also cause quality loss by producing mycotoxins, such as trichothecenes and zearalenone, which are toxic to animals and humans. For decades, chemical fungicides have been used to control FHB because of their convenience and high control efficacy. However, the prolonged use of chemical fungicides has caused adverse effects, including the emergence of drug resistance to pathogens and environmental pollution. Biological control is considered one of the most promising alternatives to chemicals and can be used for integrated management of FHB due to the rare possibility of environment pollution and reduced health risks. In this study, Bacillus velezensis JCK-7158 isolated from rice was selected as an ecofriendly alternative to chemical fungicides for the management of FHB. JCK-7158 produced the extracellular enzymes protease, chitinase, gelatinase, and cellulase; the plant growth hormone indole-3-acetic acid; and the 2,3-butanediol precursor acetoin. Moreover, JCK-7158 exhibited broad antagonistic activity against various phytopathogenic fungi and produced iturin A, surfactin, and volatile substances as active antifungal compounds. It also enhanced the expression of PR1, a known induced resistance marker gene, in transgenic Arabidopsis plants expressing ß-glucuronidase (GUS) fused with the PR1 promoter. Under greenhouse conditions, treatments with the culture broth and suspension concentrate formulation of JCK-7158 at a 1,000-fold dilution inhibited the development of FHB by 50 and 66%, respectively. In a field experiment, treatment with the suspension concentrate formulation of JCK-7158 at a 1,000-fold dilution effectively controlled the development of FHB with a control value of 55% and reduced the production of the mycotoxin nivalenol by 40%. Interestingly, treatment with JCK-7158 enhanced the expression of plant defense-related genes in salicylic acid, jasmonic acid, ethylene, and reactive oxygen species (ROS) signaling pathways before and after FHB pathogen inoculation. Taken together, our findings support that JCK-7158 has the potential to serve as a new biocontrol agent for the management of FHB.

Transcriptomic Insights into Abies koreana Drought Tolerance Conferred by Aureobasidium pullulans AK10.

The conservation of the endangered Korean fir, Abies koreana, is of critical ecological importance. In our previous study, a yeast-like fungus identified as Aureobasidium pullulans AK10, was isolated and shown to enhance drought tolerance in A. koreana seedlings. In this study, the effectiveness of Au. pullulans AK10 treatment in enhancing drought tolerance in A. koreana was confirmed. Furthermore, using transcriptome analysis, we compared A. koreana seedlings treated with Au. pullulans AK10 to untreated controls under drought conditions to elucidate the molecular responses involved in increased drought tolerance. Our findings revealed a predominance of downregulated genes in the treated seedlings, suggesting a strategic reallocation of resources to enhance stress defense. Further exploration of enriched Kyoto Encyclopedia of Genes and Genomes pathways and protein-protein interaction networks revealed significant alterations in functional systems known to fortify drought tolerance, including the terpenoid backbone biosynthesis, calcium signaling pathway, pyruvate metabolism, brassinosteroid biosynthesis, and, crucially, flavonoid biosynthesis, renowned for enhancing plant drought resistance. These findings deepen our comprehension of how AK10 biostimulation enhances the resilience of A. koreana to drought stress, marking a substantial advancement in the effort to conserve this endangered tree species through environmentally sustainable treatment.

A phenome-based functional analysis of transcription factors in the cereal head blight fungus, Fusarium graminearum.

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The fungus produces mycotoxins that are harmful to animal and human. In this study, a systematic analysis of 17 phenotypes of the mutants in 657 Fusarium graminearum genes encoding putative transcription factors (TFs) resulted in a database of over 11,000 phenotypes (phenome). This database provides comprehensive insights into how this cereal pathogen of global significance regulates traits important for growth, development, stress response, pathogenesis, and toxin production and how transcriptional regulations of these traits are interconnected. In-depth analysis of TFs involved in sexual development revealed that mutations causing defects in perithecia development frequently affect multiple other phenotypes, and the TFs associated with sexual development tend to be highly conserved in the fungal kingdom. Besides providing many new insights into understanding the function of F. graminearum TFs, this mutant library and phenome will be a valuable resource for characterizing the gene expression network in this fungus and serve as a reference for studying how different fungi have evolved to control various cellular processes at the transcriptional level.

Aureobasidium pullulans Treatment Mitigates Drought Stress in Abies koreana via Rhizosphere Microbiome Modulation.

The Korean fir tree Abies koreana, an endangered species in Korea, faces threats primarily from climate change-induced stress and drought. This study proposed a sustainable method to enhance A. koreana drought tolerance using a black yeast-like fungus identified as Aureobasidium pullulans (AK10). The 16S/ITS metabarcoding analysis assessed the impact of drought and AK10 treatment on the seedlings' rhizosphere microbiome. Results revealed a profound drought influence on the microbiome, particularly affecting fungal mycobiota. Drought-stressed seedlings exhibited elevated Agaricaceae levels, opportunistic fungi generally associated with decomposition. AK10 treatment significantly mitigated this proliferation and increased the relative abundance of beneficial fungi like Cystofilobasidium and Mortierella, known biocontrol agents and phosphate solubilizers. A notable reduction in the phytopathogenic Fusarium levels was observed with AK10, alongside an increase in beneficial bacteria, including Azospirillum and Nitrospirillum. Furthermore, the conducted correlation analysis shed light on microbial interrelationships within the rhizosphere, elucidating potential co-associations and antagonisms. Taken together, the isolated A. pullulans AK10 identified in this study serves as a potential biostimulant, enhancing the drought tolerance in A. koreana through beneficial alterations in the rhizosphere microbiome. This approach presents a promising strategy for the conservation of this endangered species.

Functional analyses of regulators of G protein signaling in Gibberella zeae.

Regulators of G protein signaling (RGS) proteins make up a highly diverse and multifunctional protein family that plays a critical role in controlling heterotrimeric G protein signaling. In this study, seven RGS genes (FgFlbA, FgFlbB, FgRgsA, FgRgsB, FgRgsB2, FgRgsC, and FgGprK) were functionally characterized in the plant pathogenic fungus, Gibberella zeae. Mutant phenotypes were observed for deletion mutants of FgRgsA and FgRgsB in vegetative growth, FgFlbB and FgRgsB in conidia morphology, FgFlbA in conidia production, FgFlbA, FgRgsB, and FgRgsC in sexual development, FgFlbA and FgRgsA in spore germination and mycotoxin production, and FgFlbA, FgRgsA, and FgRgsB in virulence. Furthermore, FgFlbA, FgRgsA, and FgRgsB acted pleiotropically, while FgFlbB and FgRgsC deletion mutants exhibited a specific defect in conidia morphology and sexual development, respectively. Amino acid substitutions in Gα subunits and overexpression of the FgFlbA gene revealed that deletion of FgFlbA and dominant active GzGPA2 mutant, gzgpa2(Q207L), had similar phenotypes in cell wall integrity, perithecia formation, mycotoxin production, and virulence, suggesting that FgFlbA may regulate asexual/sexual development, mycotoxin biosynthesis, and virulence through GzGPA2-dependent signaling in G. zeae.

Biological Control Efficacy and Action Mechanism of Klebsiella pneumoniae JCK-2201 Producing Meso-2,3-Butanediol Against Tomato Bacterial Wilt.

Bacterial wilt caused by Ralstonia solanacearum is a fatal disease that affects the production of tomatoes and many other crops worldwide. As an effective strategy to manage bacterial wilt, biological control agents using plant growth-promoting rhizobacteria (PGPR) are being developed. In this study, we screened 2,3-butanediol (BDO)-producing PGPR to control tomato bacterial wilt and investigated the action mechanism of the disease control agent. Of the 943 strains isolated from soil, Klebsiella pneumoniae strain JCK-2201 produced the highest concentration of 2,3-BDO. The culture broth of K. pneumoniae JCK-2201 did not show any direct activity on R. solanacearum in vitro, but a 100-fold dilution effectively controlled tomato bacterial wilt with a control value of 77% in vivo. Fermentation utilizing K. pneumoniae JCK-2201 was optimized to produce 48 g/L of meso-2,3-BDO, which is 50% of the sucrose conversion efficiency. In addition, the control efficacy and mechanism of meso-2,3-BDO produced by JCK-2201 in tomato bacterial wilt were determined by comparative analysis with Bacillus licheniformis DSM13 producing meso-2,3-BDO and B. licheniformis DSM13 ΔalsS that did not produce 2,3-BDO, as the step of converting pyruvate to α-acetolactate was omitted. Tomato seedlings treated with the K. pneumoniae JCK-2201 (500-fold dilution) and B. licheniformis DSM13 (100-fold dilution) culture broth produced meso-2,3-BDO that significantly reduced R. solanacearum-induced disease severity with control values of 55% and 63%, respectively. The formulated meso-2,3-BDO 9% soluble concentrate (SL; 1,000-fold dilution) showed 87% control against tomato bacterial wilt in the field condition. Klebsiella pneumoniae JCK-2201 and B. licheniformis DSM13 treatment induced the expression of plant defense marker genes, such as LePR1, LePR2, LePR5, LePR3, and PI-II, in the salicylic acid and jasmonic acid signaling pathways at 4 days after inoculation. These results show that 2,3-BDO-producing bacteria and 2,3-BDO are potential biological control agents that act through induction of resistance for controlling tomato bacterial wilt.